Method for promoting anaerobic microorganism system to efficiently produce medium-chain carboxylic acid

Abstract

The invention discloses a method for promoting an anaerobic microorganism system to efficiently produce medium-chain carboxylic acid. The nitrite is directly added into the organic wastewater, so that the microbial community structure in an anaerobic microbial system is changed, the metabolic pathway of a substrate is optimized, the chain extension process is enhanced, the production of the medium-chain carboxylic acid is promoted, the starting time of the production of the medium-chain carboxylic acid in the anaerobic system can be effectively shortened, and the yield of the medium-chain carboxylic acid is increased.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of organic wastewater resource utilization, and relates to a method for promoting efficient production of medium-chain carboxylic acid by an anaerobic microbial system. BACKGROUND

[0002] Anaerobic digestion (AD) technology is considered as an important way to realize the reuse of waste biomass resources (such as activated sludge, food waste, beer fermentation broth, etc.). However, the final product of traditional AD technology, methane biogas, has the disadvantages of single use, low value and low energy density. Compared with methane biogas, medium-chain carboxylic acid (MCCA), which is a carboxylic acid containing 6-10 carbon atoms generated by the action of carbon chain extension bacteria (CEB) on the substrate of short-chain carboxylic acid (SCCA, carboxylic acid with less than 5 carbon atoms) which is the intermediate product of AD technology, is considered to represent the future development direction of new AD technology because of its wide application prospect, high economic value and high energy density, and has therefore received close attention recently.

[0003] Using a mature and stable anaerobic microbial system as inoculum is a common strategy for constructing an anaerobic MCCA production system. A mature anaerobic microbial system often has relatively rich microbial diversity, stability and environmental adaptability, and therefore has the potential to produce MCCA. However, the microbial diversity of the anaerobic system also brings the problem of relatively low abundance of functional flora, which requires a long time of culture to complete the enrichment of the flora and the transformation of the microbial community function. In addition, potential substrate-competitive species (such as methanogens) present in the inoculated microorganisms can also affect the performance of the fermentation system, and even lead to the failure of system construction.

[0004] The application of nitrite for substrate pretreatment of anaerobic fermentation has been reported. For example, Chinese patent CN105238822A discloses a method for promoting the production of MCCA by anaerobic fermentation of residual sludge, using sludge from the secondary sedimentation tank of a municipal wastewater treatment plant as the fermentation substrate, adding nitrite to the fermentation substrate, and adjusting the pH (5-6) and temperature (20-30℃) to generate a certain amount of free nitrous acid (FNA), pretreating for 24 hours, then inoculating anaerobic sludge at a mass ratio of 1:2-2:1 of anaerobic sludge to waste activated sludge, and performing anaerobic fermentation to produce MCCA. The application of nitrite in the above method is to destroy the cell structure of the fermentation substrate (secondary sedimentation tank sludge) by the toxic effect of FNA generated from nitrite, to promote the release of organic matter, to indirectly promote the production of MCCA by increasing the concentration of organic matter, but this patent requires an additional 24h pretreatment time, increasing the overall time cost of the process. SUMMARY

[0005] The application aims to provide a method for promoting the efficient production of medium-chain carboxylic acid by an anaerobic microbial system by adding nitrite. The method directly adds nitrite to organic wastewater entering the anaerobic system, directly affects the anaerobic system through nitrite, changes the microbial community structure in the system, optimizes the substrate metabolic pathway and electron transfer process, and strengthens the chain elongation process, thereby obtaining higher yield and productivity of medium-chain carboxylic acid under the same concentration of organic matter as the prior art.

[0006] The technical solution for achieving the object of the application is as follows:

[0007] A method for promoting the efficient production of medium-chain carboxylic acid by an anaerobic microbial system, the specific steps are as follows:

[0008] (1) Add organic wastewater, nitrite and methanogenesis inhibitor to the anaerobic reactor inoculated with anaerobic sludge, adjust the initial pH value of the anaerobic reactor to 7.2-7.6, pass nitrogen to deoxygenate, and seal the reactor to prevent oxygen from entering;

[0009] (2) Start the anaerobic reactor and control the stirring speed and reaction temperature, and run in a closed mode to produce medium-chain carboxylic acid.

[0010] Further, in step (1), the organic wastewater is synthetic wastewater containing ethanol and acetic acid, food wastewater or beer fermentation liquid, etc., and the molar ratio of ethanol to acetic acid is preferably 3:1-6:1, and the ethanol concentration is preferably 110-130 mM.

[0011] Further, in step (1), the methanogenesis inhibitor is a common methanogenesis inhibitor in the art, preferably sodium 2-bromoethyl sulfonate, and the concentration is 2-4 g / L.

[0012] Further, in step (1), the initial concentration of nitrite in the anaerobic reactor is 10-20 mg N / L, preferably 20 mg N / L.

[0013] Further, in step (1), the anaerobic sludge is sludge from a stably operated anaerobic facility, which contains Clostridium, Romboutsia or Caproiciproducens and other microorganisms related to chain elongation. Preferably, the concentration of anaerobic sludge is 1.5-3 g VS / L. In the specific embodiment of the application, 2.4 g VS / L is used as an example.

[0014] Further, in step (1), the initial pH is adjusted by adding acid or base, such as hydrochloric acid or sodium hydroxide.

[0015] Further, in step (2), the reactor adopts a sequencing batch process, the reactor operation time of each batch is 6-8 days, the standing precipitation time at the end of the batch is 6-8 hours, and the drainage volume is 70-90% of the working volume.

[0016] Further, in step (2), the reaction temperature is 30-40 DEG C, the stirring is magnetic or mechanical stirring, and the rotating speed is 60-120 rpm.

[0017] The present application improves the sludge microbial community structure by adding an appropriate amount of nitrite in the organic wastewater, optimizes the substrate metabolic pathway and electron transfer process, strengthens the chain elongation process, and promotes the production of medium-chain carboxylic acid. The present application strengthens the production of MCCA in the anaerobic microbial system by adding nitrite, effectively improves the MCCA yield, optimizes the substrate metabolic pathway, improves the utilization rate and conversion rate, greatly shortens the adaptation time of the anaerobic microbial system, optimizes the MCCA production process, and narrows the gap between basic research and practical application. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The substrate and product concentration changes during the operation of CG (A), 5 mg N / L (B), 10 mg N / L (C), 15 mg N / L (D), 20 mg N / L (E) groups and the material component COD proportion (F) of each group on the 30th day of operation;

[0019] Figure 2 The COD concentration of MCCA on the 30th day (A) and the proportion of the carbon atom concentration of each material to the initial carbon atom concentration on the 30th day (B);

[0020] Figure 3 The daily concentration changes of the substrate and product during the 60-day extension experiment;

[0021] Figure 4 The proportion of the carbon atom concentration of MCCA to the initial carbon atom concentration on the last day of each cycle of the extension experiment (A), the summary of the carbon atom proportion of the final MCCA in the 7 cycles of the second three stages of the extension experiment (B), and the reactor headspace methane and hydrogen gas components (C) of the 7-10 cycles of the extension experiment;

[0022] Figure 5 The daily changes of the carbon atom proportion of MCCA in the 7 cycles of the second three stages of the extension experiment are summarized. DETAILED DESCRIPTION

[0023] The present application will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present application are not limited thereto.

[0024] The reagents used in the following examples are all available from commercial sources.

[0025] Example 1

[0026] Anaerobic sludge stored in a 4 °C refrigerator was taken out and allowed to recover to room temperature. The sludge was washed three times by centrifugation with simulated wastewater, and then the initial concentration of the anaerobic sludge was diluted to 2.4 g VS / L using simulated wastewater, followed by bottling in 150 ml anaerobic serum bottles with a working volume of 50 ml. To find the appropriate concentration, 1230 mg of sodium nitrite was dissolved in ultrapure water and made up to volume in a 100 ml volumetric flask to obtain a sodium nitrite solution with a concentration of 12.3 g / L (2.5 g N / L). Then different amounts of sodium nitrite solution (0, 0.1, 0.2, 0.3, 0.4 mL) were added dropwise to the anaerobic serum bottles to establish a control group (CG) of 0 mg N / L and four experimental groups (5, 10, 15, 20 mg N / L) of initial nitrite concentrations, with one set of triplicates for each group. The initial pH values of the CG and experimental groups were adjusted to 7.4, and then the serum bottles were purged with nitrogen gas (10 min) and immediately sealed with a butyl rubber stopper and an aluminum cap. The incubation was carried out in the dark under mesophilic conditions (35 °C constant temperature shaker, 120 rpm). Samples were taken at 0-5 d and 7, 10, 15, 30 d, and the concentrations of ethanol and carboxylic acids along the way were determined using gas chromatography.

[0027] The composition of the simulated wastewater was as follows: 5520 mg / L (120 mM) C2H5OH, 350.00 mg / L NaHCO3, 250 mg / L NH4Cl, 310 mg / L K2HPO4·3H2O, 230 mg / L KH2PO4, 330 mg / L MgCl2·6H2O, 800 mg / L NaCl, 50 mg / L CaCl2, 2 mg / L FeCl2·4H2O, 3280 mg / L (40 mM) CH3COONa, and 1 mL / L of a trace element solution. The composition of the trace element solution was: 500 mg / L MnCl2·4H2O, 50 mg / L H3BO3, 50 mg / L CoCl2·6H2O, 50 mg / L ZnCl2·7H2O, 50 mg / L CuCl2·2H2O, 50 mg / L NiCl2·6H2O, 20 mg / L Na2MoO4·2H2O, 20 mg / L Na2SeO3, 22.4 mg / L Na2WO4·2H2O. In addition, 2 g / L of the methanogenesis inhibitor sodium 2-bromoethyl sulfonate (BES) was also added to inhibit methanogens.

[0028] Figure 1The changes of product and substrate concentrations of each group within 30d of operation time are illustrated. The experimental results show that the appropriate concentration of initial nitrite significantly promotes the conversion of substrate and the production and accumulation of MCCA (C6). Specifically, in the 30d operation, the final concentration of MCCA is increased from 0.37±0.02 g COD / L of the CG group to 0.75±0.04, 1.21±0.05, 1.83±0.04, 3.88±0.05 g COD / L of the 5, 10, 15, 20 mg N / L groups, respectively, which is increased by 1.03, 2.27, 3.95, 9.48 times, and the proportion in total COD is 2.82%, 6.10%, 10.70%, 16.52%, 34.16%, respectively. Figure 1 F). Data analysis shows that the final MCCA concentration of each experimental group is significantly different from that of the control group (p<0.05) Figure 2 A). Further carbon balance analysis shows that compared with the CG group, the proportion of MCCA carbon atoms in the 20 mg N / L group is increased from 2.61% to 25.57% Figure 2 B). These results show that the anaerobic sludge cannot quickly adapt to the nutritional conditions of synthetic wastewater under the conditions of the control group, causing stagnation of substrate utilization and low efficiency of carboxylic acid production, and almost no carbon source for MCCA synthesis; however, with the continuous optimization of the initial concentration of NO2-N, the rapid start of MCCA production by anaerobic sludge is helped, which promotes the utilization of carbon sources (ethanol and acetic acid) in wastewater by sludge microorganisms, and significantly strengthens the production of MCCA.

[0029] Example 2

[0030] Two cylindrical anaerobic sequencing batch reactors (ASBR) equipped with a water bath insulation jacket and a magnetic stirrer were operated, which were the CG group with 0 mg N / L of nitrite concentration and the experimental group with 20 mg N / L of added nitrite. The reactor was a cylinder with a diameter of 14 cm and a height of 20 cm, made of organic glass, with a working volume of 2 L and a total volume of 3 L. The outer layer of the cylinder was a water bath insulation jacket with a thickness of 1.5 cm, and the temperature was controlled at 35°C. The inoculated sludge was the same as in Example 1, and after centrifugal washing of the simulated wastewater, the sludge concentration was diluted to 2.28 g VS / L. The reactor was operated in a sequencing batch mode, specifically, in a 6d operation cycle, the reactor was operated under the stirring of a magnetic stirrer with a rotation speed of 90 rpm for 17h, and then was statically precipitated for 6h, after which the water was drained and the initial pH was adjusted to 7.4, and the oxygen was removed by nitrogen blowing. The drainage volume of each cycle was 1.7 L. The total experiment lasted for 60d, with a total of 10 cycle circulations. During the experiment, daily sampling was performed, and the concentration change data of ethanol and carboxylic acid along the way were determined by gas chromatography.

[0031] Figure 3This study describes the changes in product and substrate concentrations in the CG group and the experimental group over a 60-day experimental period. During the 60-day experiment, the first three cycles (18 days) were the reactor adaptation phase, with the anaerobic sludge gradually starting up. Substrate utilization efficiency was low, and carboxylic acid production and the proportion of MCCA were relatively low. There was no significant difference in performance between the experimental and CG groups. At the end of the first three cycles, the MCCA concentrations in the CG and experimental groups were 1.11 / 0.86 g COD / L, 14.40 / 15.87 g COD / L, and 14.06 / 14.95 g COD / L, respectively. The COD proportion of MCCA increased from 14.82% / 14.87% and 21.92 / 22.85% in the first cycle to 36.31% and 39.22% in the third cycle, respectively.

[0032] Cycles 4 and 5 were the reactor adjustment phase. Addressing the issue of a high proportion of butyric acid in the total product during the adaptation phase, the ratio of ethanol to acetic acid in the simulated wastewater was adjusted in this phase. By reducing the sodium acetate concentration from 3280 mg / L in the synthetic wastewater to 1640 mg / L, the ethanol:acetic acid ratio was increased (from 3:1 to 6:1), thus benefiting the chain elongation process. At the end of this phase, the MCCA concentration in the experimental group increased by 1.64 times compared to CG, demonstrating a significant difference. Furthermore, the MCCA proportion in the experimental group was higher than during the adaptation period; the cycle with the lowest MCCA proportion in this phase (cycle 5) was 37.61% higher than the peak data in the previous phase.

[0033] The reactor reached a stable state during cycles 6-10. Considering the reduced ethanol utilization efficiency due to the lower acetic acid concentration during the adjustment phase, resulting in a higher residual ethanol content (22.53-35.30% of the initial substrate COD), the initial sodium acetate concentration in the synthesis wastewater was increased to 2460 mg / L in this phase to improve ethanol utilization. During this phase, the COD concentration of MCCA in each cycle significantly increased from 0.57-2.27 g COD / L in the CG group to 4.46-7.18 g COD / L in the experimental group, and the proportion of MCCA in the experimental group relative to CG also increased by 0.92-8.07 times.

[0034] To further compare the differences in MCCA production between the CG and experimental groups during the extended experiment, carbon flow and carbon balance were calculated for each cycle. Figure 4 As shown in Figure A, compared to CG, the carbon flux from substrate to MCCA product in the experimental group differed significantly from the second stage onwards. Over the seven cycles of the second and third stages, the average proportion of carbon atoms converted to MCCA in the experimental group was 41.76% of the initial substrate carbon atoms, while it was 12.83% in the CG group, a significant difference. Figure 4B). The conversion ratio of the experimental group fluctuated between 26.80-56.88%, while the CG continued to decline from the peak of 25.34% in the fourth cycle to 3.78% (Fig. 2B). Figure 4 A). This was due to the enrichment of methanogenic microorganisms Methanobacteriota (abundance 25.30%) in the CG, causing the carbon metabolic pathway to shift towards methanogenesis rather than chain elongation, while methanogenic microorganisms were not enriched in the experimental group (abundance 0.01%) (Fig. 2A). Figure 5 ). This result was also confirmed by the gas composition data of the reactors in cycles 7-10: during this period, the average methane production of the CG per cycle was 4.55±1.28 mL / g∙VS, while no methane was detected in the experimental group (Fig. 2B). Figure 4 C). At the same time, the average cumulative H2 production of the experimental group in the four cycles was 71.90±16.39 mL / g∙VS, which was 2.46 times that of the control group (20.80±7.78 mL / g∙VS), and higher hydrogen partial pressure was considered to be conducive to promoting the chain elongation process (Fig. 2C). Figure 4 C). Obviously, the addition of nitrite inhibited the methanogenic process and continuously directed the carbon flow towards MCCA production shift.

[0035] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not subject to the specific details of the above examples, and various simple modifications can be made to the technical solutions of the present application within the scope of the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

Claims

1. A method for promoting the efficient production of medium-chain carboxylic acids in an anaerobic microbial system, characterized in that, The specific steps are as follows: (1) Add organic wastewater, nitrite and methanogen inhibitor to the anaerobic reactor inoculated with anaerobic sludge, adjust the initial pH of the anaerobic reactor to 7.2~7.6, introduce nitrogen to remove oxygen, and seal the reactor to prevent oxygen from entering. (2) Start the anaerobic reactor and control the stirring speed and reaction temperature. Run it in a closed system to produce medium-chain carboxylic acids.

2. The method according to claim 1, characterized in that, In step (1), the organic wastewater is synthetic wastewater containing ethanol and acetic acid, food wastewater, or beer fermentation liquid.

3. The method according to claim 1, characterized in that, In organic wastewater, the molar ratio of ethanol to acetic acid is 3:1 to 6:1, and the ethanol concentration is 110 to 130 mM.

4. The method according to claim 1, characterized in that, In step (1), the methanogenic inhibitor is sodium 2-bromoethylsulfonate with a concentration of 2-4 g / L.

5. The method according to claim 1, characterized in that, In step (1), the initial concentration of nitrite in the anaerobic reactor is 10~20 mg N / L.

6. The method according to claim 1, characterized in that, In step (1), the anaerobic sludge is the sludge from a stably operating anaerobic facility, which contains microorganisms related to chain elongation, such as Clostridium, Romboutsia, or Caproiciproducens.

7. The method according to claim 1, characterized in that, In step (1), the concentration of anaerobic sludge is 1.5~3 gVS / L.

8. The method according to claim 1, characterized in that, In step (1), the initial pH is adjusted by adding acid or alkali.

9. The method according to claim 1, characterized in that, In step (2), the reactor adopts a sequencing batch process, with each batch of reactors running for 6 to 8 days, and the settling time at the end of the batch is 6 to 8 hours, and the drainage volume is 70 to 90% of the working volume.

10. The method according to claim 1, characterized in that, In step (2), the reaction temperature is 30~40℃, and the stirring is done by magnetic or mechanical stirring at a speed of 60~120 rpm.

Images